Journal: Plants

Article Title: Phytochemical Profiling and Biological Activities of Quercus sp. Galls (Oak Galls): A Systematic Review of Studies Published in the Last 5 Years

doi: 10.3390/plants12223873

Figure Lengend Snippet: Characteristics of the selected studies.

Article Snippet: [ ] , Iran, 2021 , Phytochemistry In vitro , Phytochemical screening and quantification of constituents in the hydroalcoholic QIGE. Antioxidant and antibacterial activities , QI galls—macerated with ethanol/water solvent (70/30) for 72 h , Qualitative analysis of the constituents: phytochemical tests Quantitative determination of TPC, TFC Identification and quantification of phenolic compounds in the extract: HPLC-PDA AA assay: DPPH Antibacterial activity against Bacillus pumilus (PTCC 1274), B. subtilis (ATCC 9372), S. aureus (ATCC 25923), B. cereus (PTCC 1015), K. pneumoniae (ATCC 3583), E. faecalis (ATCC 15753), E. coli (ATCC 25922), S. epidermidis (ATCC 12228), P. aeruginosa (ATCC 27852) compared to tetracycline, ampicillin, and gentamicin: disk diffusion method MIC: microdilution assay , The hydroalcoholic QIGE: - presence of alkaloids, flavonoids, tannins, saponins, and phenolic compounds (3 phenolic acids: gallic, benzoic, and caffeic acids; 2 flavonoids: rutin and quercetin) - TPC of 16.21 mg/g and TFC of 1.78 mg/g dried galls - AA: IC 50 of 47 μg/mL - high activity against E. coli , K. pneumonia , S. aureus , and S. epidermidis , with higher MIC than tetracycline.

Techniques: In Vivo, Negative Control, Positive Control, Control, Serum Assay, AST Assay, In Vitro, Emulsion, Antioxidant Activity Assay, DPPH Assay, Enzyme Inhibition Assay, Activity Assay, Viability Assay, Transmission Assay, MTT Assay, Infection, Wound Closure Assay, Colorimetric Assay, Concentration Assay, Inhibition, Acid Assay, Gene Expression, Extraction, Bacteria, Formulation, Diffusion-based Assay, Spectrophotometry, Staining, Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Over Expression, Solvent, Microdilution Assay, Flow Cytometry, XTT Assay, Reflux, SYBR Green Assay, Fluorescence, Hemolytic Assay, Functional Assay, CCK-8 Assay, Western Blot, Protein-Protein interactions, Migration, Isolation, Zeta Potential Analyzer, Ex Vivo, Suspension, UV-Vis Spectroscopy, Recombinant, In Silico, MPO Assay, Mouse Assay, Translocation Assay

Journal: International journal of cancer. Journal international du cancer

Article Title: Targeting breast cancer-initiating/stem cells with melanoma differentiation-associated gene-7/interleukin-24

doi: 10.1002/ijc.28289

Figure Lengend Snippet: Ad.mda-7 infection induces apoptosis in breast cancer-initiating/stem cells. (a) MCF-7 cells were analyzed by flow cytometry for CD44 and CD24 expression. The gates shown were used for sorting of cells with the indicated phenotypes. (b) The sorted populations, based on surface markers, were infected with Ad.mda-7 (10, 50 and 100 pfu/cell) or Ad.vec (100 pfu/cell) and after 48 hr cell proliferation was measured by MTT assay. (c and d) Cancer-initiating/stem cells from MCF-7, T47D, MDA-MB-231 and MCF-10A were infected with Ad.mda-7 (50 or 100 pfu/cell) or Ad.vec (100 pfu/cell) and after 48 hr cell proliferation was measured by MTT assay (c) and Annexin V staining assay using flow cytometry (d) measured apoptosis. MCF-7- and T47D-initiating cells were infected with Ad.mda-7 (50 or 100 pfu/cell) or Ad.vec (100 pfu/cell) and cell lysates were prepared after 48 hr. (e) MCF-7- and T47D-initiating cells were infected with Ad.mda-7 (50 or 100 pfu/cell) or Ad.vec (100 pfu/cell) and cell lysates were prepared after 48 hr. Activation of poly(ADP-ribose) polymerase-1 (PARP), caspases 3 and 7 and expression of Bcl-2 and Bax were detected by Western blotting analysis. (f) After 48-hr infection with Ad.mda-7 or Ad.vec the apoptotic index as indicative of caspase 3/7 activity in MCF-7- and T47D-initiating cells was determined using a fluorescence microscope. The apoptotic index was calculated by dividing the number of caspase 3/7 fluorescent cells by the total number of DNA-containing cells after staining with Vybrant DyeCycle Green. Data represent mean ± SD (n = 3). Asterisk (*) represents significant difference (p < 0.5) with corresponding control.

Article Snippet: After treatment caspase 3/7 activities were measured using CellPlayer™ 96-well kinetic Caspase 3/7 reagent kits following the manufacturer’s protocol (Essen Bioscience Corp. Ann Arbor, MI).

Techniques: Infection, Flow Cytometry, Expressing, MTT Assay, Annexin V Staining Assay, Activation Assay, Western Blot, Activity Assay, Fluorescence, Microscopy, Staining

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 1. DDB1 interacts with and cleaved by NS3/4A. (A) DDB1 interacts with NS3/4A in overexpression system. The 293 cells were transfected with the indicated plasmids. Coimmunoprecipitation was performed with anti-Flag or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-Flag anti-HA. The lysates were analyzed by immunoblots with anti-DDB1 or anti-HA. (B) Endogenous DDB1 interacts with NS3/4A in JFH-1 infected cells. Huh-7 cells (5 107) were mock-infected or infected with JFH-1 (Multiplicity of Infection, MOI: 0.3) for 3 days. Coimmunoprecipitation was performed with anti-DDB1 or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-DDB1 and anti-NS3. The lysates were analyzed by immunoblots with anti-DDB1 or anti-NS3.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Control, Western Blot, Infection

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 2. NS3/4A cleaves DDB1 at C378. (A) Cleavage of DDB1 by NS3/4A is inhibited by the NS3/4A inhibitor VX-950. The 293 cells were transfected with N-terminal or C-terminal Flag-tagged DDB1 (N-Flag-DDB1 or DDB1-C-Flag respectively) and HA-NS3/4A. The transfected cells were treated with VX-950 (0.2 mM) or left untreated for 1 day before immunoblot analysis with anti-Flag or anti-HA. (B) Alignment of the junction sequences of NS proteins of HCV and the potential NS3/4A cleavage sites in TC-PTP, VISA, TRIF and DDB1. (C) NS3/4A cleaves DDB1 at C378. The 293 cells were transfected with the indicated plasmids and cells lysates were analyzed by immunoblots with anti-Flag or anti-HA. (D) DDB1 N-terminal cleavage product migrated similarly to overexpressed DDB1(1–378) mutant. The 293 cells were transfected with the indicated plasmids, treated with VX-950 or left untreated for 1 day before immunoblot analysis with with anti-Flag or anti-HA.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Transfection, Western Blot, Mutagenesis

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 3. DDB1 plays a critical role in HCV replication. (A) Overexpression of DDB1 potentiates HCV RNA replication. Huh-7 cells (1 106) were transfected with the indicated amounts of Flag-DDB1 plasmid for 24 h and then cells were split and mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-Flag or anti-b-actin. Graphs show mean7SD, n¼3. (B) Knockdown of DDB1 inhibits HCV RNA replication. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (C) Knockdown of DDB1 inhibits HCV protein expression. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 3 days, and the cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (D) Knockdown of DDB1 inhibits production of infectious HCV particles. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show mean7SD, n¼3. (E) Knockdown of DDB1 inhibits RNA replication of HCV subgenomic replicon. Control or DDB1-RNAi knockdown Huh-7 cells and Huh-7 Con1 subgenomic replicon cells were cultured for 3 days. The cells (2 106) were collected and intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. Cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (F) DDB1 has no effects on HCV entry. Control or DDB1-RNAi knockdown Huh-7 cells were infected with HCVpp for 3 days (NC: Negative Control, HCVpp pakaging without HCV E1E2). The lysates of infected cells were assayed by luciferase reporter assays and immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Western Blot, Knockdown, Control, Expressing, Staining, Cell Culture, Negative Control, Luciferase

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 4. DDB1 cleavage is required for HCV replication. (A) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) The indicated stable cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh medium was added for 48 h. The JFH-1-containing medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D) Control or DDB1-RNAi knockdown Huh-7 cells were stably transduced with empty vector, DDB1, DDB1(C378R), off-target nonsense mutants of DDB1 or DDB1(C378R) respectively. Two days later, cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-actin. Graphs show meanþSD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Stable Transfection, Infection, Quantitative RT-PCR, Western Blot, Staining, Control, Knockdown, Transduction, Plasmid Preparation

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 5. DDB1 cleavage products do not affect the HCV infection. (A) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D–F) Control or DDB1-RNAi-#1 (targeted sequence is within the cDNA fragment encoding aa379–1140) transduced Huh-7 cells were further transfected with empty vector, Flag-DDB1(1–378), Flag-DDB1(379–1140*) (*, a RNAi off-target mutant),or a combination of Flag-DDB1(1–378) and Flag-DDB1(379–1140*) by Lipofectamine 2000. One day post transfection, the cells were split and mock infected or infected with JFH-1(MOI: 0.3) for 3 (D, E) or 1 (F) day. Intracellular HCV RNA levels (D), intracellular viral particles (E), or viral titers in the medium (F) were then determined as described in (A–C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Infection, Stable Transfection, Transduction, Quantitative RT-PCR, Western Blot, Staining, Control, Sequencing, Transfection, Plasmid Preparation, Mutagenesis

Journal: Cell calcium

Article Title: Ca 2+ -dependent binding of S100A6 to cofilin-1 regulates actin filament polymerization-depolymerization dynamics.

doi: 10.1016/j.ceca.2021.102457

Figure Lengend Snippet: Fig. 1. Interaction of S100A6 with cofilin-1 in NIH3T3 fibroblasts. (A) Pull-down assay with the use of protein lysate from NIH3T3 cells and S100A6 affinity resin (upper panel) or empty resin (lower panel). Lanes: 1-input, 2-unbound fraction, 3-last wash, 4-first wash with 0.5 M NaCl, 5-last wash with 0.5 M NaCl, 6-first wash with 1 M NaCl, 7- last wash with 1 M NaCl, 8- elution in buffer containing EGTA. Fractions were analyzed by SDS-PAGE (15% gel) fol lowed by immunoblotting developed with anti- cofilin-1 antibody. (B) Co-immunoprecipitation of S100A6 with cofilin-1 from NIH3T3 cell lysate. 30 μg of protein lysate was used directly for immunoblotting (input; lane 1 in both upper and lower panel) and 2.5 mg of protein lysate was incubated with (upper panel) or without (control, lower panel) anti-S100A6 monoclonal antibody and then with protein A/G agarose. In both panels, lane 2 shows unbound fraction, lane 3-last wash and lane 4-elution. Proteins were identified by immunoblotting using anti- cofilin-1 antibody. (C) Presence of S100A6- cofilin-1 complexes in NIH3T3 cells studied by PLA. Complexes of examined proteins are visualized in red; cell nuclei, stained with DAPI, are in blue. Scale bar is 20 μm.

Article Snippet: For co-immunoprecipitation assays 2.5 mg of protein lysate from NIH3T3 cells, obtained using the Plasma Membrane Protein Extraction Kit (Abcam) according to the manufacturer’s instruction was incubated with protein A/G-Agarose (Santa Cruz Biotechnology) for 1 h at 4◦C, as described by Jurewicz et al. [31].

Techniques: Pull Down Assay, SDS Page, Western Blot, Immunoprecipitation, Incubation, Control, Staining

Journal: Experimental & Molecular Medicine

Article Title: Thrap3 promotes R-loop resolution via interaction with methylated DDX5

doi: 10.1038/s12276-021-00689-6

Figure Lengend Snippet: a U2OS cells were transfected with scrambled (control) or Thrap3-targeted (Thrap3) siRNA for 24 h. The cells were then subjected to a PLA to assess the physical closeness of PCNA and R-loops. Representative images are presented. The scale bar represents 1 μm. b , c siRNA-transfected cells were transfected a second time with a plasmid expressing RNaseH1, as indicated. EdU-Click labeling was performed, and EdU incorporation was visualized. b Representative images. The white arrows indicate EdU-negative nuclei. c The Mean intensity and percentage of EdU-positive cells were measured from >30 cells. Mean ± SEM; n = 3; ** P < 0.01, *** P < 0.001, **** P < 0.0001. The scale bar represents 2 μm. d siRNA-transfected cells were transfected a second time with a plasmid expressing RNaseH1, as indicated. Cells were fixed and immunostained for γH2AX. (Left) representative images. (Right) the number of γH2AX foci per nucleus after subtraction of nucleolar signals was determined from at least 30 nuclei. Mean ± SEM, **** P < 0.0001. The scale bar represents 2 μm. e siRNA-transfected cells were transfected a second tim e with a plasmid expressing RNaseH1, as indicated. The cells were fixed and immunostained for 53BP1. (Left) representative images. (Right) the number of 53BP1 foci per nucleus after subtraction of nucleolar signals was determined from at least 30 nuclei. Mean ± SEM; ** P < 0.01, **** P < 0.0001. The scale bar represents 2 μm. f U2OS cells were transfected with scrambled (Control) or Thrap3-targeted (Thrap3) siRNA for 24 h. Cells were trypsinized, and 1000 cells were seeded in 6-well plates. The cells were allowed to proliferate for 12 days and were then fixed and stained with 10% methylene blue in 70% ethanol. The colonies were counted. (Left) representative images. (Right) the numbers of stained cell colonies per well. Mean ± SEM, ** P < 0.01. g U2OS cells were transfected with scrambled (Control) or Thrap3-targeted (Thrap3) siRNA for 24 h. Cells were trypsinized, 10 4 cells were plated in 24-well plates, and the cell confluence was monitored every 12 h using an IncuCyte FLR cell imaging microscope. Mean ± SEM; ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: To analyze the proliferation ability of MCF7 cells, 10 4 cells were seeded in a 24-well plate, and the cell number was monitored using an IncuCyte FLR cell imaging microscope (Essen Bioscience, Ann Arbor, MI, USA) every day.

Techniques: Transfection, Plasmid Preparation, Expressing, Labeling, Staining, Imaging, Microscopy

Journal: Experimental & Molecular Medicine

Article Title: Thrap3 promotes R-loop resolution via interaction with methylated DDX5

doi: 10.1038/s12276-021-00689-6

Figure Lengend Snippet: a MCF-7 cells were transfected as indicated and were then subjected to EdU-Click labeling analysis. The scale bar represents 5 μm. (Left) representative images; the white arrows indicate EdU-negative nuclei; (right) the percentage of EdU-positive cells was measured from >30 cells. Mean ± SEM, n = 3, ** P < 0.01. b Cells were transfected with scrambled siRNA (control) or Thrap3-targeted (Thrap3) siRNA for 48 h. Cells were fixed and immunostained for S9.6 and nucleolin. (Left) representative images; (right) the number of S9.6 foci per nucleus after subtraction of nucleolar signals was determined from at least 30 nuclei. Mean ± SEM, ** P < 0.01. The scale bar represents 0.5 μm. c siRNA-transfected cells were transfected a second time with a plasmid expressing RNaseH1, as indicated. Cells were fixed and immunostained for γH2AX or 53BP1. (Top) representative images; (bottom) percentages of γH2AX- and 53BP1-positive nuclei were calculated from at least 30 cells. Mean ± SEM, n = 3, ** P < 0.01, **** P < 0.0001. The scale bar represents 1 μm. d MCF-7 cells expressing scrambled (control) or Thrap3 (shThrap3) shRNA were analyzed by immunoblotting for Thrap3, DDX5, and XRN2. e MCF-7 cells expressing scrambled shRNA (control), Thrap3 shRNA #1 (shThrap3 #1), or Thrap3 shRNA #2 (shThrap3 #2) were transfected with RNaseH1 overexpression plasmids. Cells were then analyzed using an IncuCyte assay to confirm cell growth. (Top) the relative cell confluence was measured daily for a 90 h period. Mean ± SEM; n = 3; * P < 0.05, ** P < 0.01. # P < 0.05, comparison between vector- and RNaseH1-transfected cells. (Bottom) Representative cell confluency at 90 h after seeding. Mean ± SEM; n = 3; * P < 0.05, ** P < 0.01, ## P < 0.01, comparison to control cells. $ P < 0.05, comparison between vector- and RNaseH1-transfected cells.

Article Snippet: To analyze the proliferation ability of MCF7 cells, 10 4 cells were seeded in a 24-well plate, and the cell number was monitored using an IncuCyte FLR cell imaging microscope (Essen Bioscience, Ann Arbor, MI, USA) every day.

Techniques: Transfection, Labeling, Plasmid Preparation, Expressing, shRNA, Western Blot, Over Expression, Comparison

Journal: Materials Today Bio

Article Title: Lactoferrin-cyanidin-3-glucoside nanoparticles alleviate inflammation and oxidative stress via Sesn2/Nrf2 activation in mastitis

doi: 10.1016/j.mtbio.2025.102491

Figure Lengend Snippet: Schematic illustration of lactoferrin-cyanidin-3-glucoside-encapsulated nanoparticles alleviating LTA-induced inflammation and oxidative stress via Sesn2/Nrf2 activation in mastitis.

Article Snippet: Antibodies IL-1β (16806-1-AP), STAT2 (16674-1-AP), SLC7A11 (26864-1-AP), GAPDH (60004-1-Ig), Nrf2 (16396-1-AP), HO-1 (10701-1-AP), Keap1 (10503-2-AP), NQO1 (11451-1-AP), MPO (22225-1-AP), SOCS3 (14025-1-AP), and SESN2 (66297-1-Ig) were obtained from Proteintech BioTechnology (Wuhan, China).

Techniques: Activation Assay

Journal: Materials Today Bio

Article Title: Lactoferrin-cyanidin-3-glucoside nanoparticles alleviate inflammation and oxidative stress via Sesn2/Nrf2 activation in mastitis

doi: 10.1016/j.mtbio.2025.102491

Figure Lengend Snippet: Synthesis and characterization of LF-C3G-encapsulated nanoparticles. (A) Schematic illustration of the synthesis of LF-C3G-encapsulated nanoparticles (LF-C3GNPs). (B) SEM images of LF-C3GNPs (scale bar = 100 nm). (C) TEM images (scale bar = 100 nm, 200 nm). (D) Particle size distributions of LF-C3GNPs via DLS. (E) Zeta potential values to assess surface charge and colloidal stability of LF-C3GNPs. (F) UV–visible absorbance spectra of LF-C3GNPs. (G) FTIR spectrum of LF-C3GNPs. (H) The controlled release profiles of free C3G and LF-C3GNPs via UV–Vis spectroscopy. (I) Colloidal stability of LF-C3GNPs in diverse media (deionized water, DMEM, PBS, 0.9 % NaCl) by tracking hydrodynamic diameter changes over time via DLS. (J) Visual inspection and photographic image of LF-C3GNPs in different solutions. (K) Quantitative analysis of the scavenging rate of H 2 O 2 by LF-C3GNPs relative to free C3G. (L) Changes in the size of LF-C3GNPs induced by ROS (H 2 O 2 ) stimulation, as determined by DLS. (M) Schematic illustration of the H 2 O 2 -responsive mechanism of LF-C3GNPs.

Article Snippet: Antibodies IL-1β (16806-1-AP), STAT2 (16674-1-AP), SLC7A11 (26864-1-AP), GAPDH (60004-1-Ig), Nrf2 (16396-1-AP), HO-1 (10701-1-AP), Keap1 (10503-2-AP), NQO1 (11451-1-AP), MPO (22225-1-AP), SOCS3 (14025-1-AP), and SESN2 (66297-1-Ig) were obtained from Proteintech BioTechnology (Wuhan, China).

Techniques: Zeta Potential Analyzer, UV-Vis Spectroscopy

Journal: Materials Today Bio

Article Title: Lactoferrin-cyanidin-3-glucoside nanoparticles alleviate inflammation and oxidative stress via Sesn2/Nrf2 activation in mastitis

doi: 10.1016/j.mtbio.2025.102491

Figure Lengend Snippet: Effects of LF-C3GNPs on LTA-induced inflammation in HC11 cells. (A) Schematic diagram of cell culture protocol, LTA-induced inflammation in HC11 pretreated with LF-C3GNPs, the experimental verification. ( B-D) Cell viability of LF, C3G, and LF-C3GNPs. (E – J) mRNA (E – I) and protein (J) expression levels of MPO, TNF-α, IL-1β, IL-6, and SOCS3 in HC11 cells. (K) Immunofluorescence staining of TNF-α. Scale bar: 50 μm. (L – O) mRNA (L – N) and protein (O) expression levels of phosphorylated STATs (p-STAT1, p-STAT2, and p-STAT3) and STAT1, STAT2, and STAT3 in HC11 cells after different treatments. The data are presented as mean ± standard deviation (SD) from three independent replicates. Statistical significance is designated as # p<0.05 compared to the untreated control group; ∗ p<0.05 , ∗∗ p<0.01 , ∗∗∗ p<0.001 , and ∗∗∗∗ p<0.0001 relative to the LTA-treated groups.

Article Snippet: Antibodies IL-1β (16806-1-AP), STAT2 (16674-1-AP), SLC7A11 (26864-1-AP), GAPDH (60004-1-Ig), Nrf2 (16396-1-AP), HO-1 (10701-1-AP), Keap1 (10503-2-AP), NQO1 (11451-1-AP), MPO (22225-1-AP), SOCS3 (14025-1-AP), and SESN2 (66297-1-Ig) were obtained from Proteintech BioTechnology (Wuhan, China).

Techniques: Cell Culture, Expressing, Immunofluorescence, Staining, Standard Deviation, Control

Journal: Materials Today Bio

Article Title: Lactoferrin-cyanidin-3-glucoside nanoparticles alleviate inflammation and oxidative stress via Sesn2/Nrf2 activation in mastitis

doi: 10.1016/j.mtbio.2025.102491

Figure Lengend Snippet: LF-C3GNPs preferentially target STAT2/3 rather than STAT1 in LTA-stimulated cells. (A – G) Western blot and quantification showing the effects of LF-C3GNPs and STAT1 overexpression (Oe-STAT1) on STAT1/p-STAT1 and downstream inflammatory mediators (MPO, TNF-α, IL-1β, IL-6) in LTA-stimulated cells. (H) Heatmap of relative mRNA expression of genes. (I – O) Western blot and quantification demonstrating the effects of LF-C3GNPs and STAT2 overexpression (Oe-STAT2) on STAT2/p-STAT2 and downstream mediators. (P) Heatmap of relative mRNA expression of genes. (Q – W) Western blot and quantification showing the effects of LF-C3GNPs and STAT3 overexpression (Oe-STAT3) on STAT3/p-STAT3 and downstream mediators. (X) Heatmap of relative mRNA expression of genes. Data are presented as mean ± SD (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 ; ns, not significant.

Article Snippet: Antibodies IL-1β (16806-1-AP), STAT2 (16674-1-AP), SLC7A11 (26864-1-AP), GAPDH (60004-1-Ig), Nrf2 (16396-1-AP), HO-1 (10701-1-AP), Keap1 (10503-2-AP), NQO1 (11451-1-AP), MPO (22225-1-AP), SOCS3 (14025-1-AP), and SESN2 (66297-1-Ig) were obtained from Proteintech BioTechnology (Wuhan, China).

Techniques: Western Blot, Over Expression, Expressing

Journal: Materials Today Bio

Article Title: Lactoferrin-cyanidin-3-glucoside nanoparticles alleviate inflammation and oxidative stress via Sesn2/Nrf2 activation in mastitis

doi: 10.1016/j.mtbio.2025.102491

Figure Lengend Snippet: Effects of LF-C3GNPs on H 2 O 2 -induced ROS imbalance in HC11 cells. (A) ROS fluorescence images and (C) ROS quantification of HC11 cells treated with LF, C3G, and LF-C3GNPs, followed by H 2 O 2 exposure, respectively (n = 3). (B) CCK-8 assay for cell viability of LF, C3G, and LF-C3GNPs. (D, E) JC-1 staining to assess mitochondrial membrane potential (ΔΨm) (D) and corresponding fluorescence intensity ratios (red/green) across treatment groups (E) (Scale bar: 50 μm). (F, G) Lipid peroxidation visualized via C11-BODIPY 581/589 -staining (F) after different treatments and quantitative analysis of oxidative membrane damage (G) (Scale bar: 50 μm). Mean ± SD; # p<0.05 compared to the untreated control group; ∗ p<0.05 , ∗∗ p<0.01 , ∗∗∗ p<0.001 , and ∗∗∗∗ p<0.0001 relative to the H 2 O 2 -treated groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Antibodies IL-1β (16806-1-AP), STAT2 (16674-1-AP), SLC7A11 (26864-1-AP), GAPDH (60004-1-Ig), Nrf2 (16396-1-AP), HO-1 (10701-1-AP), Keap1 (10503-2-AP), NQO1 (11451-1-AP), MPO (22225-1-AP), SOCS3 (14025-1-AP), and SESN2 (66297-1-Ig) were obtained from Proteintech BioTechnology (Wuhan, China).

Techniques: Fluorescence, CCK-8 Assay, Staining, Membrane, Control

Journal: Materials Today Bio

Article Title: Lactoferrin-cyanidin-3-glucoside nanoparticles alleviate inflammation and oxidative stress via Sesn2/Nrf2 activation in mastitis

doi: 10.1016/j.mtbio.2025.102491

Figure Lengend Snippet: Effect of LF-C3GNPs on H 2 O 2 -induced oxidative stress and inflammation in HC11 cells via Sesn2/Nrf2 signaling activation. (A – D) Antioxidant enzyme activity of SOD, CAT, GPx and lipid peroxidation marker (MDA) levels in HC11 cells (n = 3). (E – L) RT-qPCR analysis (E – K) and Western blotting (L) of key redox regulators biomarkers (Nrf2, SLC7A11, Keap1, Sesn2, NQO1, HO-1, and GPX4) under different treatments (n = 3). (M – O) Immunofluorescence staining (M) and quantification (N, O) of Nrf2 and Sesn2 in HC11 (n = 3). Scale bar: 50 μm. Mean ± SD; # p<0.05 compared to the untreated control group; ∗ p<0.05 , ∗∗ p<0.01 , ∗∗∗ p<0.001 , and ∗∗∗∗ p<0.0001 relative to the H 2 O 2 -treated groups.

Article Snippet: Antibodies IL-1β (16806-1-AP), STAT2 (16674-1-AP), SLC7A11 (26864-1-AP), GAPDH (60004-1-Ig), Nrf2 (16396-1-AP), HO-1 (10701-1-AP), Keap1 (10503-2-AP), NQO1 (11451-1-AP), MPO (22225-1-AP), SOCS3 (14025-1-AP), and SESN2 (66297-1-Ig) were obtained from Proteintech BioTechnology (Wuhan, China).

Techniques: Activation Assay, Activity Assay, Marker, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Control

Journal: Materials Today Bio

Article Title: Lactoferrin-cyanidin-3-glucoside nanoparticles alleviate inflammation and oxidative stress via Sesn2/Nrf2 activation in mastitis

doi: 10.1016/j.mtbio.2025.102491

Figure Lengend Snippet: Effects of LF-C3GNPs on S. aureus -induced inflammation in mice. (A) Schematic diagram of mastitis modeling and LF-C3GNPs pre-administration in S. aureus -induced mastitis in mice. (B) H&E assay changes of S. aureus -infected mammary tissues in different groups. Scale bar: 500 μm. (C) Bacterial colony-forming unit (CFU) on agar plates and (D) quantification of CFU after different treatment groups. (E) Heatmap quantification and (F) proteins expression levels of MPO, TNF-α, IL-1β, IL-6, and SOCS3 in infected mice CFU after different treatment groups. (G) Immunofluorescence staining and (H) quantification of TNF-α. Scale bar: 200 μm. (I – K) mRNA and (L) Protein expression levels of phosphorylated STATs (p-STAT1, p-STAT2, and p-STAT3) and STAT1, STAT2, and STAT3 in infected mice. Mean ± SD; # p<0.05 compared to the untreated control group; ∗ p<0.05 , ∗∗ p<0.01 , ∗∗∗ p<0.001 , and ∗∗∗∗ p<0.0001 relative to the S. aureus -treated groups.

Article Snippet: Antibodies IL-1β (16806-1-AP), STAT2 (16674-1-AP), SLC7A11 (26864-1-AP), GAPDH (60004-1-Ig), Nrf2 (16396-1-AP), HO-1 (10701-1-AP), Keap1 (10503-2-AP), NQO1 (11451-1-AP), MPO (22225-1-AP), SOCS3 (14025-1-AP), and SESN2 (66297-1-Ig) were obtained from Proteintech BioTechnology (Wuhan, China).

Techniques: Infection, Expressing, Immunofluorescence, Staining, Control

Journal: Materials Today Bio

Article Title: Lactoferrin-cyanidin-3-glucoside nanoparticles alleviate inflammation and oxidative stress via Sesn2/Nrf2 activation in mastitis

doi: 10.1016/j.mtbio.2025.102491

Figure Lengend Snippet: Effect of LF-C3GNPs on S. aureus -induced oxidative stress and inflammation in mice via Sesn2/Nrf2 signaling activation . (A – D) Antioxidant enzyme activity of SOD, CAT, GPx and lipid peroxidation marker (MDA) levels in S. aureus -infected mammary tissue (n = 3). (E – L) Western blotting (E) and RT-qPCR analysis (F – L) of key redox regulators biomarkers (Nrf2, Sesn2, Keap1, SLC7A11, NQO1, HO-1, and GPX4) under different treatments (n = 3). (M – O) Immunofluorescence staining (M) and quantification (N, O) of Nrf2 and Sesn2 in S. aureus -infected mammary tissue (n = 3). Scale bar: 50 μm. Mean ± SD; # p<0.05 compared to the untreated control group; ∗ p<0.05 , ∗∗ p<0.01 , ∗∗∗ p<0.001 , and ∗∗∗∗ p<0.0001 relative to the S. aureus -treated groups.

Article Snippet: Antibodies IL-1β (16806-1-AP), STAT2 (16674-1-AP), SLC7A11 (26864-1-AP), GAPDH (60004-1-Ig), Nrf2 (16396-1-AP), HO-1 (10701-1-AP), Keap1 (10503-2-AP), NQO1 (11451-1-AP), MPO (22225-1-AP), SOCS3 (14025-1-AP), and SESN2 (66297-1-Ig) were obtained from Proteintech BioTechnology (Wuhan, China).

Techniques: Activation Assay, Activity Assay, Marker, Infection, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining, Control

Journal:

Article Title: Identification of Ebp1 as a component of cytoplasmic bcl - 2 mRNP (messenger ribonucleoprotein particle) complexes

doi: 10.1042/BJ20051548

Figure Lengend Snippet: (A) RNA gel-shift assay. Aliquots of the indicated fractions were incubated with 32P-AREbcl-2 RNA in RNA binding buffer. Free and bound RNAs were separated by electrophoresis on a 0.8% agarose gel, which was dried and analysed by phosphorimaging. (B) and (C), aliquots of the indicated fractions were incubated with 32P-AREbcl-2 RNA in RNA binding buffer and exposed to UV light for 30 min. Complexes were digested with RNase A and T1, and proteins were separated by SDS/PAGE. (B) Image of the Coomassie Blue stained gel. (C) Phosphorimage of the same SDS gel. MW, molecular mass markers; S, sample applied to the column; FT, flow through fraction; W, wash fraction.

Article Snippet: Briefly, 5 μl of 32 P-ARE bcl - 2 RNA transcripts (2×10 5 c.p.m.) was incubated with 10 μl of the concentrated column fraction in RNA-binding buffer (without BSA) at 4 °C for 10 min followed by UV irradiation (254 nm) for 30 min. After UV cross-linking, reactions were treated with RNase A (Ambion, Inc.) (0.01 units/μl) and RNase T1 (0.4 units/μl) at 37 °C for 30 min.

Techniques: Electrophoretic Mobility Shift Assay, Incubation, RNA Binding Assay, Electrophoresis, Agarose Gel Electrophoresis, SDS Page, Staining, SDS-Gel

Journal:

Article Title: Identification of Ebp1 as a component of cytoplasmic bcl - 2 mRNP (messenger ribonucleoprotein particle) complexes

doi: 10.1042/BJ20051548

Figure Lengend Snippet: Heparin–sepharose fraction 3 was incubated with ARE-RNA-poly(A):oligo(dT) beads. Proteins were eluted from the RNA affinity matrix with a step gradient of NaCl. Eluted fractions were examined by gel-shift and UV cross-linking assays. (A) RNA gel-shift assays of column fractions. Aliquots of the fractions were incubated with 32P-AREbcl-2 RNA in binding buffer and RNA–protein complexes were separated by electrophoresis on a 0.8% agarose gel. (B) Aliquots of the indicated fractions were separated by SDS/PAGE and detected by Coomassie Blue staining. (C) Aliquots of the fractions eluted with 0.4 and 0.6 M NaCl were incubated with 32P-AREbcl-2 RNA and exposed to UV light. Complexes were digested with RNase A and RNase T1 and proteins were separated by SDS/PAGE. Bands were detected by phosphorimaging of the SDS gel. MW, molecular mass markers; S, sample applied to the column; FT, flow through fraction; W, wash fraction. Arrows indicate the bands excised from the gel for MALDI-MS analysis.

Article Snippet: Briefly, 5 μl of 32 P-ARE bcl - 2 RNA transcripts (2×10 5 c.p.m.) was incubated with 10 μl of the concentrated column fraction in RNA-binding buffer (without BSA) at 4 °C for 10 min followed by UV irradiation (254 nm) for 30 min. After UV cross-linking, reactions were treated with RNase A (Ambion, Inc.) (0.01 units/μl) and RNase T1 (0.4 units/μl) at 37 °C for 30 min.

Techniques: Incubation, Electrophoretic Mobility Shift Assay, Binding Assay, Electrophoresis, Agarose Gel Electrophoresis, SDS Page, Staining, SDS-Gel

Journal:

Article Title: Identification of Ebp1 as a component of cytoplasmic bcl - 2 mRNP (messenger ribonucleoprotein particle) complexes

doi: 10.1042/BJ20051548

Figure Lengend Snippet: Cytosolic extracts were incubated with monoclonal anti-nucleolin or control anti-(mouse IgG) antibodies and protein complexes were recovered as described in Figure 6. The presence of Ebp1 in immunoprecipitates (IP) was determined by Western blotting using a polyclonal anti-Ebp1 antibody. Pulldown reactions were carried out using an anti-nucleolin antibody (lane 2), an anti-nucleolin antibody after pre-treatment with RNase A for 15 min (lane 3), or an anti-(mouse IgG) antibody (negative control) (lane 4). Total extracts (input control) were analysed in lanes 1 and 6. Supernatants (Sup) from the precipitation reactions were analysed in lanes 7–9 as indicated.

Article Snippet: Briefly, 5 μl of 32 P-ARE bcl - 2 RNA transcripts (2×10 5 c.p.m.) was incubated with 10 μl of the concentrated column fraction in RNA-binding buffer (without BSA) at 4 °C for 10 min followed by UV irradiation (254 nm) for 30 min. After UV cross-linking, reactions were treated with RNase A (Ambion, Inc.) (0.01 units/μl) and RNase T1 (0.4 units/μl) at 37 °C for 30 min.

Techniques: Incubation, Western Blot, Negative Control

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Data from AlphaFold 3, predicting (A) NAT10 dimerization and the degree of interaction between (B) NAT10-DDB2, (C) NAT10-p53, and (D) DDB2-p53, were imported to ChimeraX 1.9 software showing individual proteins. NAT10 is highlighted by orange, DDB2 is green, p53 is blue colour. The source data are shown at https://www.ibp.cz/en/research/departments/cellular-biology-and-epigenetics/open-data . This raw data from AlphaFold 3 can be uploaded to ChimeraX 1.9 for more detailed analysis.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Software

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: (A) The following proteins were studied in non-irradiated and UVC-irradiated NAT10 wt and dn cells: NAT10, p53, XPA, XPC, and DDB2. In detail, panel (A) shows western blot analysis of proteins NAT10, p53, XPA, XPC, and DDB2 in non-irradiated and UVC-irradiated cells without and with a depletion of the NAT10 acetyltransferase. Data revealed lower p53, XPA, XPC, and DDB2 protein levels in NAT10 dn cells, with further reductions of XPA, XPC, and DDB2 protein levels upon UVC exposure. UVC irradiation also decreased the pool of p53, XPA, and XPC in NAT10 wt cells. Panels (B-D) show the quantification of western blot data from panel (A) using ImageJ software (NIH freeware, USA). Non-irradiated cells and cells irradiated by UVC light were analyzed. (E) TP53 deficiency led to a decrease in the pool of NAT10 and DDB2 proteins. Panel (F) shows the quantification of western blot data from panel (E) using ImageJ software (NIH freeware, USA). The protein levels were normalized to GAPDH (reference and loading control). The asterisks in panels B-D and F represent statistically significant differences with either a p-value ≤ 0.05 (*) or a p-value ≤ 0.001 (***). AlphaFold 3 models of ( G) NAT10 interaction with p53 (pTM = 0.53), ( H ) NAT10 and DDB2 (pTM = 0.5), and ( I ) DDB2 and p53 protein (pTM = 0.47). Structures are colored with AlphaFold 3 pLDDT confidence score.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Irradiation, Western Blot, Software, Control

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Panels (A, B) show AlphaFold 3 multimer prediction of human NAT10 interaction with (A) NAT10 and p53 and DDB2 proteins (pTM = 0.48) or (B) NAT10 and p53 and DDB1-DDB2 protein complex complex (pTM = 0.53).

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques:

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Interaction properties between p53-DDB2 and NAT10-DDB2 in human NAT10 wt and NAT10 dn cells were studied using PLA. (A) The panel shows an example of PLA methodology, adapted from BioRender software. In (B) NAT10 wt and (C) NAT10 dn cells, after fixation, the PLA was performed using antibodies to detect the p53-DDB2 or NAT10-DDB2 complexes in both non-irradiated and UVC-irradiated NAT10 wt and NAT10 dn cells. Red dots indicate amplified interaction signals, with cell nuclei counterstained with DAPI, used for visualization of DNA content. NAT10-DDB2 in NAT10 dn acted as a control, showing no external impact on PLA outcomes. This section quantifies PLA signals (dots per nucleus) for (D) the p53-DDB2 complex in non-irradiated and UVC-irradiated NAT10 wt cells, (E) p53-DDB2 in non-irradiated and UVC-irradiated NAT10 dn cells. (F) The UVC irradiation significantly increased NAT10-DDB2 PLA signals in NAT10 wt cells, suggesting enhanced interaction. Non-irradiated cells and cells irradiated by UVC light were analyzed. We used ImageJ software (NIH freeware, USA) to analyze the number of PLA signals. The statistical analysis was performed using GraphPad Prism 9 software (USA) and the nonparametric Mann-Whitney U -test. The asterisk in panel F represents statistically significant differences with a p-value ≤ 0.05 (*).

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Software, Irradiation, Amplification, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: (A, B) Animation documents chromatin density inside the cell nucleus (see graphical illustration created by BioRender software). Panel (A) shows cell nucleolus (Nu) and nuclear lamina in dark blue, and panel (B) shows localization of heterochromatin (green) decorating the periphery of cell nucleus and nucleolus, while euchromatin (pale blue) is shown as de-condensed treads inside the cell nucleus. An analysis of the DDB2 nuclear distribution pattern was done using immunohistochemistry in (C) NAT10 wt and NAT10 dn cells. (D) The NAT10 protein (red) was accumulated into tiny, well-visible foci in DAPI-dense (considered as heterochromatin), DAPI-poor (euchromatin, more decondensed) genomic region as well as inside compartment of nucleoli. Panel (E) shows representative images of the DDB2 protein recruitment to UVA-microirradiated chromatin in NAT10 wt and NAT10 dn cells. Microirradiation using a 405 nm laser line was performed, followed by immunofluorescent staining for DDB2 and DNA damage marker γH2A.X. DAPI was used for nuclear counterstaining in blue fluorescence. The scale bar represents 7.5 μm.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Software, Immunohistochemistry, Staining, Marker, Fluorescence

Journal: Cell Death Discovery

Article Title: Mitochondrial priming and response to BH3 mimetics in “one-two punch” senogenic-senolytic strategies

doi: 10.1038/s41420-025-02379-y

Figure Lengend Snippet: A Top . Representative images ( n = 3) of SA-β-gal staining in A549 lung cancer cells treated with senescence-inducing concentrations of bleomycin, alisertib, doxorubicin or palbociclib for 7 days (scale bar: 500 μm for proliferative/untreated cells; 200 μm for TIS cells). Bottom left . Expression levels of phospho-RB Ser807/Ser811 and p21 WAF1/Cip1 were detected by immunoblotting in whole cell lysates of proliferative (untreated) and TIS A549 cancer cells (7-day) using specific antibodies and β-actin/GAPDH as loading controls. The figure shows a representative immunoblot from multiple ( n = 3) independent experiments (PLB Palbociclib, DOX Doxorubicin, ALI Alisertib, BLEO Bleomycin). Bottom right . Representative flow cytometry plots showing the gating of the cell cycle distribution of proliferative (untreated) and TIS phenotypes. The histogram shows the percentage (mean ± S.D., n = 3) of cells in the four cell cycle phases as a function of the treatment condition. B Top . Representative microphotographs of proliferative (untreated) and TIS cancer cells labeled with IncuCyte ® Annexin V Green reagent 3 days after harvest and reseeding. Bottom . TIS cancer cells and proliferative (untreated) cells were reseeded into 96-well plates (6000 cells/well and 2000 cells/well, respectively) and cultured with increasing concentrations of ABT-263/navitoclax. After 5 days, cell viability was measured using the alarmarBlue™ assay. Shown are representative images of alamarBlue™-based cell viability assays showing changes in the number of metabolically active cells in response to serial dilutions of BH3 mimetics and dose-response curves for ABT-263/navitoclax in proliferative (untreated) and TIS A549 lung cancer cells (untreated = 100%). Bar graphs of the IC 50 values determined as the μmol/L concentrations of ABT-263/navitoclax required to decrease cell viability by 50%, and the senolytic indexes obtained by dividing the IC 50 values of ABT-263/navitoclax in proliferative A549 cells by those obtained in TIS A549 cells. Data represent the mean ± S.D. of ≥3 independent experiments performed in triplicate. Statistically significant differences (ANOVA analysis) between proliferative and TIS phenotypes means are shown. n.s. not statistically significant. C TIS cancer cells and proliferative (untreated) cells were replated into 96-well plates (6000 cells/well and 2000 cells/well, respectively) and cultured with increasing concentrations of ABT-199/venetoclax, A1331852 or S63845. After 5 days, cell viability was measured using the alarmarBlue™ assay. Shown are representative images of alamarBlue™-based cell viability assays showing changes in the number of metabolically active cells in response to serial dilutions of BH3 mimetics and dose-response curves for ABT-199/venetoclax, A1331852 or S63845 in proliferative (untreated) and TIS A549 lung cancer cells (untreated = 100%). See also Table .

Article Snippet: Incucyte ® Annexin V Dye for apoptosis (Cat. #4642) was purchased from Sartorius (Göttingen, Germany).

Techniques: Staining, Expressing, Western Blot, Flow Cytometry, Labeling, Cell Culture, Metabolic Labelling

Journal: Cell Death Discovery

Article Title: Mitochondrial priming and response to BH3 mimetics in “one-two punch” senogenic-senolytic strategies

doi: 10.1038/s41420-025-02379-y

Figure Lengend Snippet: A Top . Both the level of mitochondrial priming—the proximity to the mitochondrial apoptotic threshold that determines the ability of TIS mitochondria to initiate apoptosis in response to BH3 peptides—and the nature of apoptotic blockade in TIS cancer cells—the distinct patterns of dependence on pro-survival BCL-2 proteins in the BCL2/BH3 interactome—were assessed using the plate-based JC-1 BH3 profiling. After proliferative (untreated) and TIS cancer cells are permeabilized with digitonin to allow BH3 peptides to diffuse into the cells and interact with intact mitochondria, the loss of JC-1 red fluorescence caused by depolarization of the mitochondrial transmembrane potential (a surrogate for the endpoint MOMP) allows real-time kinetic measurements of the loss of mitochondrial integrity. Primed mitochondria respond more robustly to both activator and sensitizer BH3 peptides and are more susceptible to apoptosis. The pattern of response to BH3 peptides can also identify the functional dynamics between pro- and anti-apoptotic proteins in maintaining cell survival in the TIS phenotypes (e.g., defects in pro-apoptotic signaling or increased addiction to anti-apoptotic proteins). Cell viability analysis using a panel of BH3 mimetics (ABT-263, ABT-199, A13318512, S63845) was used to assess senolytic indexes (SI), which were defined as the ratio of IC 50 values of proliferative cancer cells to IC 50 values of TIS cancer cells. Bottom . Apoptotic blocks based on BH3 profiling. After exposure to individual activator or sensitizer BH3 peptides, BH3 profiling can distinguish three major blocks (stop signs) through which cells can avoid apoptosis, namely: primed, unprimed-competent, and unprimed-incompetent. Bottom . Patterns of interaction between the anti-apoptotic proteins present in cells ( columns ) and the pro-apoptotic synthetic peptides or drugs ( rows ) used in the BH3 profiling assay and the BH3 mimetics cell viability toolkit. BIM, BID, and PUMA inhibit all the inhibitors and are pan-sensitizers. PUMA (shown in red rows) can act as an activator of BAX and BAK. Orange colors highlight those peptides/drugs that inhibit the BCL-2 protein, including BMF and BAD, Blue rows highlight the dependency on mantle cell lymphoma (MCL)-1, whereas NOXA inhibits MCL-1 and BFL-1. Green indicates BCL-X L dependency, as HRK is primarily a BCL-X L inhibitor, but can also inhibit other anti-apoptotic proteins with lower affinity ( A and B , created with Biorender.com ) . BH3 profiling was performed to measure mitochondrial depolarization in proliferative (untreated) and TIS A549 lung cancer cells exposed to activator ( B ) and sensitizer ( C ) BH3 peptides. Figure shows heat maps of % mitochondrial depolarization caused by increasing concentrations of the activator (BIM, BID, PUMA) and sensitizer (BMF, BAD, NOXA, HRK) peptides in proliferative (untreated) and palbociclib, doxorubicin, alisertib, and bleomycin TIS cells. Data shown are the mean of ≥3 independent experiments using three technical replicates for each peptide. Graphs represent the means ( columns ) ± S.E.M. ( bars ) of ≥3 independent experiments BH3 peptide EC 50s (μmol/L) in proliferative and palbociclib, doxorubicin, alisertib, and bleomycin TIS cells. Statistically significant differences (ANOVA analysis) between proliferative and TIS phenotypes means are shown. n.s. not statistically significant. D Global heat maps of % mitochondrial depolarization caused by selected concentrations of activator and sensitizer peptides in A549 lung cancer cells. Samples are ordered according to depolarization by the HRK peptide. Data shown are the mean of ≥3 independent experiments with three technical replicates for each peptide.

Article Snippet: Incucyte ® Annexin V Dye for apoptosis (Cat. #4642) was purchased from Sartorius (Göttingen, Germany).

Techniques: Fluorescence, Functional Assay